The spectrum of cardiometabolic disease encompasses the Metabolic Syndrome, Pre-diabetes, Type 2 Diabetes (T2DM), and cardiovascular disease, and these diseases are responsible for a huge burden of patient suffering and health care costs among veterans and the US population in general. Insulin resistance at the level of skeletal muscle is central to the underlying pathogenesis of all these disease manifestations. Regarding the mechanism of insulin resistance, the conventional hypothesis is that oxidation of long-chain fatty acids (LCFA) is impaired while availability is increased, and this results in the accumulation of intramyocellular lipid (IMCL) and metabolites of LCFAs such as diacylglycerol, LCFA-CoAs, and ceramides. These latter compounds activate various serine kinases, such as PKC or IKK2 (in association with activation of NF-:B), which then phosphorylate insulin receptor substrate (IRS) molecules resulting in impaired insulin signal transduction. However, our preliminary data point to an alternative or complementary pathophysiological process. The first clue was that IMCL was completely independent of insulin sensitivity in African Americans, while oxidative stress related to lipid peroxidation in muscle was predictive of insulin resistance in both European- and African-Americans. The second clue was provided by metabolomic studies of plasma obtained from normal controls and insulin-resistant patients with T2DM. The metabolomic profile in T2DM was consistent with incomplete oxidation of long-chain fatty acids (LCFA) as evidenced by accumulation of medium chain acylcarnitines, rather than increased LCFAs and accumulation as IMCL triglyceride, together with decreased tri-carboxylic acid (TCA) cycle activity and diminished anaplerosis. Given these observations, we wish to test a novel hypothesis that the abnormal metabolome is the result of intrinsic mitochondrial defects that both impair FA oxidation and generate reactive oxygen species (ROS). Activation of serine kinases is then mediated either directly by medium-chain acylcarnitines and/or by peroxidized lipids generated by mitochondrial ROS that activate inflammatory pathways (e.g., NG-:B). In this proposal, we will test these hypotheses in humans. Normoglycemic insulin sensitive and insulin resistant subjects, and patients with T2DM will be metabolically characterized by hyperinsulinemic clamps, body composition, IMCL, energy expenditure and substrate oxidation rates, and will be studied before and after perturbations that alter insulin sensitivity including short-term very low calorie diet, after stabilization following 10% weight loss, and insulin-sensitizing thiazolidinedione treatment. Metabolomic profiles will be assessed for acylcarnitines, fatty acids, organic acids, amino acids, and LCFA-CoAs in plasma, urine, and in biopsied muscle tissue. In addition, mitochondria isolated from muscle will be functionally studied for substrate oxidation capacity using high resolution respirometry, reactive oxygen species generation, and activity of individual respiratory complexes. Oxidative stress will be measured in muscle by hydroxyl-nonenal and protein carbonyls. The effect of oxidative stress and/or metabolomic analytes to activate inflammatory pathways in muscle leading to serine phosphorylation and desensitization of IRS and insulin signaling will be examined. These studies represent a state-of-the-art application of new metabolomics technologies, combined with molecular studies of mitochondrial function and insulin signaling, in human muscle, and will test new hypotheses regarding the link between abnormal lipid metabolism and insulin resistance. These data will predictably identify new diagnostic biomarkers for insulin resistance and T2DM, and advance our understanding of molecular mechanisms underlying human insulin resistance.